Methylphenidate (MPD) and amphetamine (AMPH) are stimulants of abuse that are used by millions for the treatment of attention deficit hyperactivity disorder. Our laboratory recently identified previously unreported effects of these agents. Specifically, MPD rapidly and reversibly increases vesicular dopamine (DA) uptake in a vesicle-enriched subcellular fraction prepared from treated rats. In contrast, AMPH analogs decrease vesicular DA uptake in the same fraction. These effects occur concurrent with increases and decreases in vesicular monoamine transporter-2 (VMAT-2) immunoreactivity after MPD and AMPH analog treatment, respectively, and appear to reflect trafficking of VMAT-2 protein and/or vesicles within nerve terminals. Accordingly, this proposal will test the hypothesis that MPD affects vesicular DA transport and the intraneuronal localization of VMAT-2 in a manner distinct from AMPH. This will be accomplished by: A. demonstrating the unique features of MPD on VMAT-2 localization, and comparing these properties with those of AMPH. Specifically, we will: i) identify the vesicles associated with the redistribution of VMAT- 2, and the subcellular compartments among which VMAT-2 are trafficking; ii) determine the regional selectivity of the MPD-induced redistribution of VMAT-2, and if this change is unique to DA neurons; and iii) determine the impact of age and chronic treatment on the MPD-induced redistribution of VMAT-2. B. elucidating mechanisms whereby MPD alters VMAT-2 localization. This will be accomplished by investigating: i) the role of D1 and D2 receptors in redistributing VMAT-2; ii) the acute effect of MPD on D2 receptor localization; and iii) the role of synapsin in the MPD-induced changes in VMAT-2 distribution. C. demonstrating functional consequences of the effect of MPD on vesicular trafficking. Specifically, we will investigate if: i) MPD alters vesicular DA sequestration; and ii) MPD-induced alterations in VMAT-2 distribution are neuroprotective against the long-term monoaminergic damage caused by high-dose methamphetamine administration. Completion of these studies will advance our understanding of the therapeutic effects of MPD and AMPH, as well as their abuse and dissimilar neurotoxic liabilities. These data will also provide insight into mechanisms underlying the regulation of vesicular DA uptake and sequestration. Finally, these studies will advance our understanding of a wide array of processes including those underlying addiction and those contributing to the development of DA neurodegenerative diseases.